Callitriche Cophocarpa (Water Starwort) Proteome Under Chromate Stress: Evidence for Induction of a Quinone Reductase
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Environmental Science and Pollution Research (2018) 25:8928–8942 https://doi.org/10.1007/s11356-017-1067-y RESEARCH ARTICLE Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase Paweł Kaszycki1 & Aleksandra Dubicka-Lisowska1 & Joanna Augustynowicz2 & Barbara Piwowarczyk2 & Wojciech Wesołowski3 Received: 16 May 2017 /Accepted: 18 December 2017 /Published online: 13 January 2018 # The Author(s) 2018. This article is an open access publication Abstract Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) → Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress. Keywords Macrophyte . Aquatic plants . Hexavalent chromium . Biological reduction . Phytoremediation . Oxidative stress . Quinone dehydrogenase Responsible editor: Elena Maestri Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-1067-y) contains supplementary material, which is available to authorized users. Anthropogenic pollution with Cr compounds has become a worldwide problem due to extensive use of this metal in a * Paweł Kaszycki number of industrial applications and vehicular transport [email protected] (Shadreck and Mugadza 2013; Singh et al. 2013; Zayed and Terry 2003). Chromium may appear at various oxidation 1 Unit of Biochemistry, Institute of Plant Biology and Biotechnology, states forming chemical agents of different toxicities, solubil- Faculty of Biotechnology and Horticulture, University of Agriculture ities, and stabilities (Kotaś and Stasicka 2000; Zayed and in Kraków, al. 29 Listopada 54, 31-425 Kraków, Poland Terry 2003), and among these, chromate (Cr(VI)) is the most 2 Unit of Botany and Plant Physiology, Institute of Plant Biology and hazardous to living organisms (Saha et al. 2011;Zhitkovich Biotechnology, Faculty of Biotechnology and Horticulture, 2− 2011). Chromate ion (CrO4 ) structurally resembles the sul- University of Agriculture in Kraków, al. 29 Listopada 54, fate anion (SO 2−) and therefore it becomes actively incorpo- 31-425 Kraków, Poland 4 rated by cells mainly through non-specific sulfate transporter 3 Unit of Genetics, Plant Breeding and Seed Science, Institute of Plant systems and in the lesser extent through HPO 2− carriers Biology and Biotechnology, Faculty of Biotechnology and 4 Horticulture, University of Agriculture in Kraków, al. 29 Listopada (Cervantes et al. 2001; Prado et al. 2016; Singh et al. 2013). 54, 31-425 Kraków, Poland Then, inside cells, Cr(VI) as a strong oxidant interacts with Environ Sci Pollut Res (2018) 25:8928–8942 8929 cell constituents and undergoes rapid reduction due to both knowledge on Cr bioconversion mechanisms and response enzymatic and non-enzymatic reactions (Cervantes et al. strategies. 2001; Chandra and Kulshreshtha 2004; Shanker et al. 2005, The mechanisms of chromate uptake, accumulation, and 2009). Chromate bioreduction is in fact a gradual and complex metabolism have been studied for terrestrial plants more thor- route involving formation of Cr(V) and Cr(IV) intermediates oughly than for macrophytes (Shanker et al. 2005, 2009), responsible for generation of reactive oxygen species (ROS). although the latter group seems to be a better choice for In plants, many of these byproducts lead to adverse stress phytoremediation purposes (Tel-Or and Forni 2011). This is effects by causing severe physiological disorders and oxida- because aquatic plants, especially the water-floating and sub- tive damage (Oliveira 2012; Panda and Choudhury 2005; merged species, usually exhibit higher accumulation potential Singh et al. 2013). and have evolved distinct physiological and biochemical path- For the above reasons, Cr decontamination actions need to ways due to their larger contact area with the surrounding be undertaken. Among various methods implemented to date water environment (Chandra and Kulshreshtha 2004). In con- environmentally, bioremediation and especially sequence, the assimilative organs of macrophytes are affected phytoremediation appear as the most promising approaches directly by the solution and this interaction becomes particu- (Parvaiz 2016). In biotechnological applications, plants capa- larly important in the case of heavy metal presence. ble of enhanced chromate uptake and accumulation as well as Several aquatic plants have been proposed as efficient of its reduction to the less toxic forms are of particular interest phytoremediators of Cr contamination (Prado et al. 2016; since they bring possibilities for elaborating cheap, non-inva- Singh et al. 2013, and the references therein). Among these sive, and efficient industrial-scale methods for soil reclama- is the recently studied genus Callitriche that appears as a suit- tion and water cleanup (Prado et al. 2016;Yangetal.2005; able candidate for environmental biotechnological use Zayed and Terry 2003). However, biological remediation of (Augustynowicz et al. 2014b; Favas et al. 2012, 2014). In environmental contamination with chromium compounds in- particular, Callitriche cophocarpa Sendtn. (water starwort), volves complex and divergent processes (Chandra and a widespread species growing both in stagnant and running Kulshreshtha 2004; Panda and Choudhury 2005;Shanker waters, was shown to reveal unusual response to chromate et al. 2005, 2009). In the case of higher plants, chromium proving to be a potent Cr phytoremediator and accumulator stress negatively affects cellular metabolism at different func- (Augustynowicz et al. 2010, 2013a, 2013b, 2016). It exhibited tional levels triggering variant reactions that enable some spe- enhanced capability of both Cr(VI) and Cr(III) cies to adapt to this heavy metal, resist its action, or detoxify phytoextraction from contaminated waters and extremely high hazardous intermediates (Prado et al. 2016; Shanker et al. capacity to bind Cr (3900 mg kg−1 for 5-day treatment at 2009; Zayed and Terry 2003). 1 mM Cr(VI)). Furthermore, it revealed extraordinary poten- Chromate detoxication and/or protective mechanisms in tial in terms of chromate accumulation rate (up to 1.8% of dry plants are complex, may involve multiple factors, and can mass which makes it a Cr hyperaccumulator), intratissular become manifested at different organizational levels related bioreduction kinetics (a postulated detoxication pathway), to plant metabolism, physiology, development, or structure. and bioconcentration factor (BCF determined as 74 for a 5- For the above reasons, they are not yet fully explained and day treatment of the shoots with 1 mM Cr(VI), the value close understood (see Prado et al. 2016 for a recent critical review). to that of commercially used sorbents). Upon incubation with Moreover, these processes differ depending on the strategy 50-μMchromate,C. cophocarpa accumulated > 0.1% of Cr used by particular species to cope with the metal toxicity. with no noticeable changes in the plant physiological status. Based on these divergent strategies, plants can be categorized All the above characteristics imply that the macrophyte has as Cr excluders, indicators, or accumulators (Dalvi and evolved efficient adaptive mechanisms against chromate Bhalerao 2013). For the case of Cr-accumulating and Cr- stress and can respond to Cr(VI) treatment atypically. tolerant plants, it is well evidenced that the chromate stress So far, little has been done with regard to the studies of reaction involves induction of oxidative stress response sys- chromate-induced response of macrophytes at the level of tems. This is achieved by enhancing the activities of antioxi- detectable proteomes. Such research is of high interest, espe- dant enzymes (superoxide dismutase (SOD), catalase (CAT), cially for the case of submerged plants that can interact with peroxidase, glutathione reductase, ascorbate peroxidase, Cr compounds